Process for purification of fusion protein

Abstract

The present invention is directed to the use of anion exchange chromatography to produce a CTLA4-Ig fusion protein with improve glycan.

1 . A process of purifying a CTLA4-Ig fusion protein mixture, the purification process comprising:
 a. loading the CTLA4-Ig fusion protein mixture onto anion exchange column with suitable buffer at suitable pH selected from pH 7.0 to 7.5;   b. optionally, the wash is performed;   c. eluting the CTLA4-Ig fusion protein mixture from anion exchange column;   wherein the CTLA4-Ig fusion protein mixture has low or reduce high mannose by at least 50%.   
     
     
         2 . The process as claimed in  claim 1 , wherein the CTLA4-Ig fusion protein mixture has afucosylation reduce by at least 20%. 
     
     
         3 . The process as claimed in  claim 1 , wherein the high mannose is reduced about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, and about 80%. 
     
     
         4 . The process as claimed in  claim 1 , wherein the fusion protein has afucosylation reduced about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, and about 30%. 
     
     
         5 . The process as claimed in  claim 1 , wherein process further comprises affinity chromatography, mixed mode chromatography, hydrophobic interaction chromatography and cation exchange chromatography performed before or after anion exchange chromatography. 
     
     
         6 . The process as claimed in  claim 5 , wherein the anion exchange is selected from Poros XQ, Poros HQ DEAE, Sepharose fast flow, Fractogel® EMD DEAE (M), Toyopearl DEAE-650, Toyopearl DEAE-650, Nuvia Q. 
     
     
         7 . The process as claimed in  claim 6 , wherein the Poros XQ anion exchange is strong anion exchange performs in bind elute mode comprising (i) loading the fusion protein mixture at suitable pH and/or conductivity for binding (ii) performing washing at suitable pH and/or conductivity (iii) eluting the fusion protein mixture at suitable pH and/or conductivity. 
     
     
         8 . The process in as claimed in  claim 7 , wherein the loading of fusion protein mixture at suitable pH selected from about 7.0 to about 7.5 and conductivity selected from about 5 mS/cm to about 8 mS/cm. 
     
     
         9 . The process as claimed in  claim 8 , wherein the suitable pH and/or conductivity is maintained with suitable buffer selected from sodium phosphate, Tris-HCl, Sodium citrate, and Sodium chloride. 
     
     
         10 . The process as claimed in  claim 1 , wherein the suitable washing comprises:
 (i) first wash with equilibration buffer at suitable pH 7.2±0.2 and/or conductivity 2.7±0.3 mS/cm;   (ii) second wash at the same pH as of the first wash buffer and/or a conductivity higher than the first wash buffer selected from about 5 mS/cm to about 20 mS/cm.   
     
     
         11 . The process as claimed in  claim 10 , wherein the second wash buffer comprises sodium phosphate and sodium chloride, wherein the concentration of Sodium phosphate (NaP) selected from about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and about 100 mM and Sodium chloride (NaCl) selected from about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and about 100 mM. 
     
     
         12 . The process as claimed in  claim 1 , wherein the elution is performed at suitable pH 7.2±0.2 and conductivity 30.0±3.0 mS/cm. 
     
     
         13 . The process as claimed in  claim 12 , wherein the elution is performed with buffer sodium phosphate and sodium chloride. 
     
     
         14 . The process as claimed in  claim 13 , wherein the elution is performed in linear gradient wherein the concentration of buffer increased from about 20% to about 80%, preferably about 20% to about 70% for column volume selected from about 1CV, about 2CV, about 3CV, about 4CV, about 5CV, about 6CV, about 7CV, about 8CV, about 9CV, about 10CV, about 11CV, about 12CV, about 13CV, about 14CV, about 15CV, about 16CV, about 17CV, about 18CV, about 19CV and about 20CV. 
     
     
         15 . The process as claimed in  claim 14 , wherein the elution starts, and fractions collected at an ascending value of about 10 mAU/cm and descending value of about 80 mAU/cm. 
     
     
         16 . The process as claimed in  claim 1 , the anion exchange chromatography performed in bind and elute mode. 
     
     
         17 . The process as claimed in  claim 9 , wherein the concentration of suitable buffer selected from about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1M, about 1.2M, and about 1.5M. 
     
     
         18 . The process as claimed in  claim 10 , wherein the equilibration buffer comprises Sodium phosphate (NaP), the concentration selected from about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM. 
     
     
         19 . The process as claimed in  claim 13 , wherein the elution buffer comprises Sodium phosphate (NaP) concentration selected from about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and about 100 mM and sodium chloride (NaCl) concentration selected from about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1M, about 1.2M, and about 1.5M. 
     
     
         20 . A pharmaceutical composition of CTLA4-Ig fusion protein comprises high mannose in range selected from about 0.05% to about 0.3%, afucosylation in range selected from about 0.5% to about 3%, and galactosylation in range selected from about 25% to about 38% detected by Hydrophilic interaction liquid chromatography (HILIC).